Detection of specific nucleic acids in a sample has found many applications. One of these applications is the detection of single nucleotide substitutions in genes. Single nucleotide substitutions are the cause of a significant number of inherited diseases and/or may confer a greater susceptibility to display a certain phenotype such as a disease or an infliction. Detection of nucleic acid sequences derived from a large variety of viruses, parasites and other micro-organisms is very important in medicine, the food industry, agriculture and other areas.
The relative quantification of specific nucleic acid sequences has important applications but is more complex and is therefore not routinely performed. One application of the relative quantification of DNA sequences is detection of trisomies such as Down's syndromes which is due to a trisomy of chromosome 21. In cancer cells deletions or amplifications of specific chromosomal areas often occur. Analysis of these can provide important information needed for optimal treatment. One example is amplification of the ERBB2 (Her-Neu) region on human chromosome 17 which defines a specific class of breast tumors requiring treatment different from other breast cancers. Detection of mutations as well as deleted or amplified chromosomal area's can potentially be used to distinguish benign and malignant tumors in small micro-biopts and can provide a fingerprint of a tumor for clonality analysis. Relative quantification of mRNAs is studied for many different reasons among which improved classification and molecular characterisation of tumors. Relative quantification of cytokine mRNAs from in vitro stimulated blood samples can potentially be used to characterise immune responses.
Many methods are known for the detection of specific nucleic acids in a sample. The most sensitive methods currently available rely on exponential amplification of the nucleic acid(s) to be detected e.g. with the use of the Polymerase Chain Reaction (PCR), Ligase Chain Reaction (LCR) or the self-sustained sequence amplification (3SR).
In PCR, nucleic acid oligomers are provided to the sample to enable priming of nucleic acid synthesis on specific sites on the nucleic acid. Subsequently the nucleic acid sequence between the two amplification primers is amplified through successive denaturation, hybridisation and nucleic acid polymerisation steps.
Detection of an amplified nucleic acid, a so-called amplicon, can occur in many different ways. Non-limiting examples are size fractionation on a gel followed by visualisation of nucleic acid. Alternatively, specific amplified sequence can be detected using a probe specific for a part of the amplified sequence.
When it is not, or only superficially, known what sequences to look for in a sample, it is advantageous to use a strategy in which a large variety of different sequences can be detected in a single test. When this so-called multiplex amplification is used to determine the relative abundance of various target nucleic acid in the original sample, it is particularly important that the difference in the number of amplified molecules per amplicon is correlated to the difference in the number of target sequences per amplicon in the sample.
To ensure this correlation, a bias in the amplification of sequences not due to a difference in the relative abundance of target nucleic acids in the sample should be avoided as much as possible.
Multiplex nucleic acid amplification methods can be divided in methods in which one amplification primer pair is used for all fragments to be amplified such as RAPD, AFLP and differential display techniques, and methods using a different amplification primer pair for each fragment to be amplified. The currently available amplification techniques using only one primer pair for all fragments to be amplified are typically used to amplify a random subset of the nucleic acid fragments present in a sample. It is not uncommon that more than 50 fragments are amplified in one reaction using these techniques. It has been shown by Vos et al.(1995), Nucleic Acid Research 23, 4407-14 that the Polymerase Chain Reaction as used in AFLP is capable of amplifying large numbers of unrelated fragments with almost equal efficiency provided that these fragments can be amplified with the same set of PCR primers. Relative amounts of amplification products obtained by AFLP can be used to determine relative copy number of specific fragment sequences between samples.
Multiplex methods for the amplification of specific targets typically use a different primer pair for each target sequence to be amplified. The difference in annealing efficiency of different primer pairs result in a strong bias in the amplification of the different amplicons thereby strongly reducing the fidelity of a quantitative multiplex assay. Furthermore the presence of a large number of different primers results in a strongly increased risk of primer dimer formation diminishing the possibility of reproducible amplifying small amounts of target nucleic acids. Amplification of more than 10 specific nucleic acid fragments in one test is therefore not recommended in the art and usually leads to unreliable results.
The method of the preamble is known from e.g. WO 96/15271 (herein incorporated by reference), providing a method for copying and detecting sequence information of a target nucleic acid present in a sample, into a well characterised DNA template. The method comprises hybridising up to 5 different probe sets of single stranded first and second DNA probes to a target nucleic acid wherein the first and second probe, after hybridisation to the target sequence and subsequently ligation of the probes are used as a template for amplification. The method is suited for the copying of sequence information of RNA or DNA into a DNA template. Said first and/or said second probe further comprises a tag which is essentially non-complementary to said target nucleic acid. The tags are used for the priming of nucleic acid synthesis in the amplification reaction. Such tag can also be used for detection of the resulting amplicon. Thus, said amplification is initiated by binding of a nucleic acid primer specific for said tag. A bias due to difference in primer sequences is avoided by including into the copying action a nucleic acid tag to which amplification primers are directed. Thus, for the analysis of nucleic acid in a sample the sample is provided with one or more DNA probes wherein said probes comprise a first nucleic acid tag and a second nucleic acid tag, optionally denaturing nucleic acid in said sample, incubating said sample to allow hybridisation of complementary nucleic acid in said sample, functionally separating hybridised probes from non-hybridised probes, providing said hybridised probes with at least a first primer, complementary to said first tag, and a second oligomer primer, complementary to said second tag, amplifying at least part of said DNA probes after hybridisation and analysing the amplificate for the presence of amplified products.
Said first and said second probe can only be amplified exponentially by e.g. PCR when the probes are connected. Since connection can essentially only take place when the probes are substantially adjacent to each other, exponential amplification, and thereby detection of the amplicon is only possible if said first and said second probe where hybridised to the target nucleic acid. Non hybridised probes are not exponentially amplified. Removal of non-hybridised and non-ligated probes is therefore not essential, and the reactions can be carried out in the same reaction vessel. Dependent on the temperature, buffer-conditions, ligase-enzyme and oligonucleotides used, the difference in ligation efficiency of oligonucleotides that are perfectly matched to the target nucleic acid and mismatched oligonucleotides can be very large providing increased possibilities to discriminate closely related target sequences.
A similar method is known from WO 97/45559. Both prior art methods however suffer from serious limitations preventing their use for the detection and relative quantification of more than 5 specific nucleic acid target sequences in a single “one-tube” assay in an easy to perform and robust test with unequivocal results using only a small amount of a nucleic acid sample.
The above identified prior art methods were derived from the Ligase Chain Reaction (LCR; Barany F., Proc.Natl.Acad.Sci.USA, 88:189-93 (1991). In fact, these previous art methods are designed to use two consecutive amplification reactions, starting with several cycles of LCR. In LCR very short hybridisation reactions and therefore high probe concentrations are used. The ligation and amplification reactions are performed in the same reaction vessel, i.e. without sample immobilisation and without removal of non-ligated probe molecules and buffer constituents. All probe oligonucleotides used in the ligation reaction remain therefore present during the amplification reaction. One of the tags used for amplification which is present at the 3′ end of one of the two probe oligonucleotides is however complementary to one of the PCR primers and will therefore provide a template for primer elongation during the PCR reaction. These unligated probe molecules only contain one of the two tags used in the PCR reaction and can therefore not be amplified exponentially but only linearly. During each PCR cycle each picomole of probe will consume one picomole of one of the PCR primers. For each probe pair present, the probe amounts used in the art, 200-500 femtomoles (WO97/45559) of each probe, 750-1500 femtomoles (WO96/15271) or 160 fmoles (WO 98/04746) will consume 5-45 picomoles of one of the PCR primers during the 25-30 PCR cycles that are needed when nanogram amounts of human nucleic acids are being analysed. The use of more than 10 probes simultaneously requires, apart from the amounts necessary for exponential amplification of ligated probes, PCR primer amounts in excess of 50 pMoles for the linear amplification of unligated probes (that are not removed, but still present in the reaction mixture) which results in strongly increased amounts of aspecific amplification products. The multiplex methods in the art are therefore limited to the use of a maximum of 5-10 probes per detection reaction. In related previous art methods even higher probe concentrations are used. In WO 98/37230, 5000 femtomoles of each of three probe oligonucleotides is used. In WO 97/19193, 3200 femtomoles probe are used in each assay. These previous art methods are therefore not suitable for multiplex detection of several probes. The high probe amounts used in the previous art reduces the number of probes that can be used simultaneously as well as the sensitivity of the assay.